P614: NOVEL SPLICE-SITE MUTATION IN PLCG2 ASSOCIATED WITH RESISTANCE TO BTK INHIBITORS
Authors: Jialei Qi, Martin Wist, Eugen Tausch, Deyan Yordanov Yosifov, Anja Schade, Xiang Gao, Rashmi Priyadharshini Dheenadayalan, Christof Schneider, Sascha Endres, Daniel Mertens, Peter Gierschik, Stephan Stilgenbauer, Billy Michael Chelliah Jebaraj
Journal: HemaSphere (2023)

Background: Bruton tyrosine kinase (BTK) inhibitors (BTKi) are successful for the treatment of B-cell neoplasms, but a subset of patients acquire BTK and/or phospholipase C gamma 2 (PLCG2) mutations and relapse during therapy. Splice-site mutations in PLCG2 leading to enzyme activation upon exposure to cold temperatures have been reported in PLAID syndrome (Ombrello MJ. et al., NEJM, 2012). However, their association with response to BTKi is unknown. Aims: We aimed to identify and characterize splice-site alterations in PLCG2 selected under BTKi treatment using an in vitro REC-1 cell line model. Methods: We generated resistance to a panel of covalent and non-covalent BTKi in REC-1 cell lines using a dose escalation scheme (Qi J. et al., Blood Adv, 2023). CellTiter-Glo® was used to study treatment responses to BTKi. Next-generation sequencing covering the splice-donor and acceptor sites of PLCG2, along with a panel of recurrently mutated CLL genes was performed to identify resistance associated mutations. Inositol phosphate formation assay was used to measure lipase activity of mutant and wildtype (WT) PLCγ2. RNA sequencing and gene set enrichment analysis (GSEA) were performed to identify differentially expressed pathways in PLCG2 mutants compared to WT. Results: Among the multiple parallel lines of REC-1 cells where resistance to the covalent BTKi: ibrutinib and tirabrutinib, and the non-covalent BTKi: nemtabrutinib, pirtobrutinib, vecabrutinib, fenebrutinib and RN-486 was generated, a novel PLCG2 c.2236-1G>T mutation at the splice site was observed in 2 of 3 tirabrutinib resistant lines with variant allele frequencies (VAF) of 42% and 44%, respectively. The 3rd tirabrutinib resistant line had a BTK C481F mutation with a VAF of 35%. Sanger sequencing of cDNA from PLCG2 mutant REC-1 cDNA showed a deletion of exon 21 (Δ21) in PLCG2. Western blotting analysis indicated a shorter (presumably mutant) in addition to the larger (WT) PLCγ2 protein band. Of note, exon 21 of PLCG2 encodes the BTK dependent Tyr 753 and 759 phospho-sites, important for the regulation of PLCγ2 auto-inhibition. Correspondingly, the shortened mutant band was not detected in western blot upon stimulation with anti-IgM when using the phospho-Tyr 759 antibody. In contrast, both the mutant and WT bands were detected when using the phospho-Tyr 1217 antibody. In inositol formation assay, deletion of exon 21 was found to markedly enhance lipase activity of PLCγ2 at 37°C. Further, we examined whether the Δ21 mutant displayed enhanced lipase activity at sub-physiological temperatures as known for the PLAID associated Δ19 PLCγ2 mutant. Unlike PLCγ2 Δ19, Δ21 displayed no cold-induced activation. Cell viability assay demonstrated that REC-1 cells with PLCG2 Δ21, similar to those with L854F PLCG2 mutation, were resistant to all covalent and noncovalent BTKi, with nemtabrutinib showing the highest potency (Fig. 1). RNA-seq and gene set enrichment analysis (GSEA) revealed enriched nuclear factor kappa B (NF-κB) and NOD-like receptor (NLR) consistent with active PLCγ2 mediated signaling. Summary/Conclusion: Here, we identified a novel PLCG2 splice-site mutation in BTKi resistant REC-1 cells, which led to the deletion of exon 21, most likely impairing the auto-inhibitory regulation of the enzyme and increasing lipase activity of PLCγ2. Similar to other known PLCγ2 mutants, the novel Δ21 PLCγ2 mutant showed resistance to all BTKi tested, due to BTK independent activation of BCR signaling. Our study highlights the importance of sequencing splice sites of PLCG2 in the context of patients who progress under BTKi-based treatments.Keywords: Bruton’s tyrosine kinase inhibitor (BTKi), Alternative splicing, B-CLL, Drug resistance